- Open Access
Pharmacological characterization of cnidarian extracts from the Caribbean Sea: evaluation of anti-snake venom and antitumor properties
- Cláudia S. Oliveira1, 2, 3,
- Cleópatra A. S. Caldeira1, 2, 3,
- Rafaela Diniz-Sousa1, 2, 3,
- Dolores L. Romero4,
- Silvana Marcussi5,
- Laura A. Moura6,
- André L. Fuly6,
- Cicília de Carvalho7,
- Walter L. G. Cavalcante7, 8,
- Márcia Gallacci7,
- Maeli Dal Pai9,
- Juliana P. Zuliani1, 2, 3,
- Leonardo A. Calderon1, 2, 3 and
- Andreimar M. Soares1, 2, 3, 10Email author
© The Author(s). 2018
- Received: 19 December 2017
- Accepted: 7 August 2018
- Published: 28 August 2018
Cnidarians produce toxins, which are composed of different polypeptides that induce pharmacological effects of biotechnological interest, such as antitumor, antiophidic and anti-clotting activities. This study aimed to evaluate toxicological activities and potential as antitumor and antiophidic agents contained in total extracts from five cnidarians: Millepora alcicornis, Stichodactyla helianthus, Plexaura homomalla, Bartholomea annulata and Condylactis gigantea (total and body wall).
The cnidarian extracts were evaluated by electrophoresis and for their phospholipase, proteolytic, hemorrhagic, coagulant, fibrinogenolytic, neuromuscular blocking, muscle-damaging, edema-inducing and cytotoxic activities.
All cnidarian extracts showed indirect hemolytic activity, but only S. helianthus induced direct hemolysis and neurotoxic effect. However, the hydrolysis of NBD-PC, a PLA2 substrate, was presented only by the C. gigantea (body wall) and S. helianthus. The extracts from P. homomalla and S. helianthus induced edema, while only C. gigantea and S. helianthus showed intensified myotoxic activity. The proteolytic activity upon casein and fibrinogen was presented mainly by B. annulata extract and all were unable to induce hemorrhage or fibrinogen coagulation. Cnidarian extracts were able to neutralize clotting induced by Bothrops jararacussu snake venom, except M. alcicornis. All cnidarian extracts were able to inhibit hemorrhagic activity induced by Bothrops moojeni venom. Only the C. gigantea (body wall) inhibited thrombin-induced coagulation. All cnidarian extracts showed antitumor effect against Jurkat cells, of which C. gigantea (body wall) and S. helianthus were the most active; however, only C. gigantea (body wall) and M. alcicornis were active against B16F10 cells.
The cnidarian extracts analyzed showed relevant in vitro inhibitory potential over the activities induced by Bothrops venoms; these results may contribute to elucidate the possible mechanisms of interaction between cnidarian extracts and snake venoms.
- Caribbean sea cnidarians
- Natural products
Marine organisms, which comprise half of the total global biodiversity, have been recognized as the largest remaining reservoir of novel compounds to be evaluated for drug activity [1–6]. Animals belonging the phylum Cnidaria are of great importance for studies of pharmacological and toxicological assessments. The composition of cnidarian venoms remains incompletely elucidated. However, several of their compounds have been described, including peptides, proteins, purines, quaternary ammonium compounds, biogenic amines and betaines [1, 7–11].
Venoms from such animals as snakes [12–14], scorpions [15–18], anurans [19, 20], cone snails [21, 22] and cnidarians [23–25] have been used as a source of bioactive compounds for the prospection of lead compounds potentially useful for the development of new anticancer therapies [26, 27]. This fact has provoked a growing worldwide interest in the screening of proteins, peptides, marine natural products (MNPs) from cnidarians in order to discover new anticancer bioactive compounds [28, 29].
The use of genomic and proteomic approaches had permitted a rapid increase in the number of sequences from cnidarians deposited in protein and gene databases [30–32]. Some of these toxins have been used for the development of anticancer molecules. One interesting example is the hemolytic toxin (HT) from Stichodactyla helianthus sea anemone which was conjugated with an antibody towards an antigen expressed on immature T lymphocytes (IOR-T6) producing an o-hemolytic hybrid IOR-T6-HT that showed toxicity against CEM cells expressing the IOR-T6 antigen and non-toxic effects for K562 cells without the antigen .
Additionally, several marine natural products are able to inhibit the toxic effects of snake venoms, such as extracts from Plocamium braziliense , Canistrocarpus cervicornis  and seaweed Prasiola crispa . The marine extracts that also inhibit PLA2 activity include manoalide , vidalols, and a group of terpenoids that contain masked 1,4-dicarbonyl moieties. Furthermore, the biotechnological potential of PLA2 inhibitors may provide therapeutic molecular models that exert antiophidian activity to supplement the conventional serum therapy against these multifunctional enzymes [38, 39].
This study aimed to evaluate toxicological activities and their efficacy against tumor and snake-venom toxic activities from five Caribbean Sea cnidarian species of the hydrozoa class: Millepora alcicornis, Plexaura homomalla and Cnidarians of the anthozoa class: Condylactis gigantea (total and body wall), Stichodactyla helianthus, and Bartholomea annulata.
Materials and reagents
The synthetic fluorescent substrates Acyl 6:0 NBD phospholipids, NBD-phosphatidylcholine (PC) and NBD-phosphatidic acid (PA) were purchased from Avanti Polar Lipids Inc. (USA). The reagents used in the electrophoresis, salts and other reagents were obtained from Sigma Chemical Company (USA).
The cnidarians specimens were collected in the coast of Havana City during a one-year period. The extracts of corals were obtained as previously described by , whereas anemone extracts were obtained according to . Protein quantitation was based on the Bradford method (BioRad) using bovine serum albumin (BSA) as a standard.
Adult male mice weighing 25 to 30 g were maintained under a 12 h light-dark cycle (lights on at 07:00 h) in a temperature-controlled environment (22 ± 2 °C) for at least ten days prior to the experiments. Food and water were freely available. Animal procedures were in accordance with the guidelines prepared by the Committee on Care and Use of Laboratory Animal Resources, National Research Council, USA. The ethical aspects related to the project were approved by the Ethics Committee on Animal Use (No. 2012/1) and the Ethics Committee (102/2009) for Research on Human Beings from Brazil (CAAE: 14204413.5.0000.0011).
SDS-PAGE 12.5% (m/v) was carried out as previously described . 500 μg samples C. gigantea (body-wall), C. gigantea (total), M. alcicornes, S. helianthus, P. homomalla and B. annulata were pretreated in reducing conditions (SDS plus β-mercaptoethanol) at 100 °C for 5 min. Gels were stained with 0.1% Coomassie brilliant blue R-350 in ethanol: acetic acid (5:1, v/v) for 15 min and discolored in 10% acetic acid. The molecular mass was estimated by interpolation from a linear logarithmic plot of relative molecular mass versus distance of migration using standard molar mass markers (SDS7 Sigma-Aldrich).
The Phospholipase A2 (PLA2) activity was measured using the indirect hemolytic assay on agarose gels containing red blood cells and egg yolk phospholipids . The hemolytic activity was evaluated spectrophotometrically using suspensions of fresh human RBC (red blood cells) as previously described [44, 45].
PLA2 activity was evaluated also through the hydrolysis of synthetic fluorescent phospholipid, using the fluorescent substrate Acyl 6:0 NBD phospholipid, NBD-phosphatidylcholine (NBD-PC). The assay was performed using a spectrofluorimeter (Shimadzu, RF-5301PC, software RFPC) with excitation and emission wavelengths of 460 and 534 nm, respectively. The enzymatic activity of each cnidarian extract was evaluated for 250 s after the addition of substrate (3.3 μg/mL, final concentration) in a reaction medium containing 50 mM Tris-HCl, and 8 mM CaCl2, pH 7.5, at room temperature.
Proteolytic activity assay
Proteolytic activity upon fibrinogen was measured as described by  with some modifications. Fibrinogen (70 μg) diluted in PBS was incubated with different amounts of cnidarian extracts diluted in 20 μL buffer (pH 7.5) at 37 °C for 2 h. The reaction was stopped with 20 μL of a solution containing 10% (v/v) glycerol, 10% (v/v) β-mercaptoethanol, 2% (v/v) SDS, and 0.05% (w/v) bromophenol blue. Fibrinogen hydrolysis was demonstrated by SDS-PAGE using 12% polyacrylamide gels. Proteolytic activity upon casein was measured as described by . Cnidarian extracts (100 and 500 μg) were incubated for 30 min at 37 °C in a solution of 0.1 M Tris-HCl pH 9.0 containing 1% casein. After the incubation period, 1.5 mL of 30% TCA was added to each sample to stop the enzymatic reaction and centrifuged at 340 x g for 25 min. Then, the samples were read on a spectrophotometer at a wavelength of 280 nm. One unit of protease activity was defined as the amount of enzyme that produces an increase in absorbance of 0.001 units/minute at 280 nm.
Hemorrhagic activity assay
Hemorrhagic activity was quantitatively estimated by the method of  with some modifications. Groups of six Swiss mice (18-22 g) were shaved on the back and then intradermally (i.d.) injected with different doses of cnidarians extracts or snake venoms, in 50 μL of phosphate buffered saline (PBS). After 2 h, animals were anesthetized and euthanized. The shaved back skin was removed and the hemorrhagic halo diameter was measured. The minimum hemorrhagic dose (MHD) was obtained from the mean of these diameters (mm). The MHD is defined as the dose of snake venom or extract that produces a hemorrhagic lesion of 10 mm diameter after 2 h.
Coagulant activity assay
The clotting time was determined by mixing 20 μL of the samples (in 0.15 M NaCl, pH 7.4) with 200 μL of citrated bovine plasma at 37 °C. The B. jararacussu snake venom (20 μg) was assayed in order to determine the minimum coagulant dose (corresponding to the time between 1 and 1.2 s – 100% activity). For the neutralization trials, the snake venom was previously incubated with different cnidarian extracts for 30 min at 37 °C, at different proportions (1:5, 1:10 and 1:30, w/w).
Mice were euthanized by exsanguination after previous cervical dislocation. Phrenic-diaphragm (PD) preparation was removed and mounted vertically in a conventional isolated organ-bath chamber containing 15 mL of physiological solution of the following composition (mmol/L): NaCl, 135; KCl, 5; MgCl2, 1; CaCl2, 2; NaHCO3, 15; Na2HPO4, 1; glucose, 11. This solution was bubbled with carbogen (95% O2 and 5% CO2). The preparation was attached to an isometric force transducer (Grass, FT03) for recording the twitch tension. The transducer signal output was amplified and recorded on a computer via a transducer signal conditioner (Gould, 13–6615-50) with an Acquire Lab Data Acquisition System (Gould). The resting tension was 5 g; indirect contractions were stimulated by supramaximal pulses (0.2 Hz, 0.5 ms) delivered from an electronic stimulator (Grass-S88 K) and applied to the phrenic nerve by means of a suction electrode. The preparation was allowed to stabilize for 45 min before the addition of a single concentration of toxin .
At the end of the myographic study, the diaphragm muscle was removed from the bath and frozen in liquid nitrogen. Transverse sections (8 mm thick) were cut out at − 20 °C in a cryostat and stained with hematoxylin and eosin (HE) prior to examination by light microscopy . Muscle damage was quantified in HE stained preparations, using an Analysis Imaging System (Leica, Qwin). The number of fibers with lesions was expressed as a percentage of the total number of cells (muscle damage index), in three non-overlapping non-adjacent areas of each muscle, observed at the same magnification.
Creatine kinase release
The creatine kinase (CK) assay was carried out using the CK-UV kinetic kit from Sigma Chem. Co. Different cnidarian extracts were injected (i.m., 50 μL) into Swiss male mice weighing 18–22 g (n = 6). The control animals received 0.15 M PBS. After 3 h, the blood from the tail was collected in heparin-coated tubes and centrifuged for plasma separation. The amount of CK was then determined using 4 μL of plasma, which was incubated for 3 min at 37 °C with 1.0 mL of the reagent. Enzyme activity was expressed in international units per liter (IU/L), with one unit of activity corresponding to phosphorylation of 1 μmol of creatine/min at 25 °C.
Edema inducing activity
Groups of six Swiss male mice (18–22 g) were injected in the sub plantar region with different doses of cnidarian extracts in 50 μL of PBS. After 0.5, 1 and 3 h, the paw edema was measured using a low-pressure spring caliper (Mytutoyo-japan) [51, 52]. The zero time values were then subtracted and the differences reported as median % ± S.D.
Tumor cell cytotoxic activity of cnidarian extracts on human acute T-cell leukemia (Jurkat) and B16F10 cell lines were assayed using the MTT method according to . Cells were dispersed in 96-well plates at a density of 1 × 105 cells per well. After 24 h of culture, the media were removed and fresh media, with or without different concentrations of samples, were added into the wells and incubated for 24 h. The extracts were evaluated at 1000, 100 and 10 μg/mL concentrations using Vincristine as positive control (100 μg/mL). Results were expressed as a percentage (%).
Results were expressed as mean ± S.D. Data was analyzed by ANOVA complemented by the Tukey-Kramer test, using the statistical program GraphPad 5.0. Values of p < 0.05 were considered significant.
However, fractions enriched with these two molecules together demonstrated a significant increase in phospholipase activity . Although CgPLA2 and Sticholysin I and II are known to exert phospholipase activity, it should be emphasized that the experiments were carried out with total extract. It is possible that these molecules are responsible for the hydrolysis promoted against the NBD-PC substrate, but we do not rule out the existence of other molecules that are components of the extract, which alone or in clusters may be acting in the hydrolysis of the NBD-PC substrate.
The extracts of C. gigantea (body wall) and B. annulata were able to partially hydrolyze the α and β chains of fibrinogen; however, S. helianthus, M. alcicornis and P. homomalla were incapable of hydrolyze fibrinogen efficiently (Fig. 1b). The fibrinogenolytic assay was carried out using 50 μg of the cnidarian extracts, whose proteolytic activity upon casein was evaluated; furthermore, the extract of B. annulata (100 μg) hydrolyzed casein at 80 U/min. The extracts of C. gigantea (body wall) and B. annulata at 500 μg hydrolyzed casein at 118 and 170 U/min, respectively (Fig. 2c).
Clotting activity inhibition from B. jararacussu snake venom by cnidarian extracts
Clotting Time (min.)
B. jararacussu (20 μg)
C. gigantea (body-wall)
C. gigantea (total)
At 3 h after the injection of 50 μg of cnidarian extracts into the gastrocnemius mouse muscle, a slight myotoxic effect from the extracts of M. alcicornis and P. homomalla was observed, approximately 22% above those observed for the controls injected with PBS alone. The C. gigantea (body-wall), C. gigantea (total) and S. helianthus presented increased activity, while extract of B. annulata did not show myotoxic effect.
The neutralization of the clotting induced by B. jararacussu snake venom and the inhibition of the hemorrhagic activity induced by B. moojeni venom were demonstrated by the majority of the cnidarian extracts tested, whereas the ability to inhibit thrombin-induced coagulation was shown by the C. gigantea (body wall). Together with the anti-tumor effect against JURKAT cells demonstrated by all cnidarian extracts tested and the specificity shown against B16F10 cells, these findings constitute important evidence that cnidarians extracts are a rich source of bioactive molecules that should be studied in order to produce data for the development of new alternatives for snakebite envenomation and cancer therapies.
This work was supported by Ministry of Science and Technology (MCTI), National Council for Scientific and Technological Development (CNPq), Funding Authority for Studies and Projects (FINEP), Coordination for the Improvement of Higher Education Personnel (CAPES), National Research Network in Marine Biotechnology (process no. 408522/2013-5, MCTI/CNPq/FNDCT) and Rondônia Research Foundation (FAPERO). The authors express their gratitude to Genetic Heritage Management Board (CGEN/MMA) for financial support under the authorization number 010627/2011-1. The authors thank the Program for Technological Development in Tools for Health-PDTIS-FIOCRUZ for use of its facilities. Amy Grabner provided the English editing of the manuscript. The authors thank the Andrea A. Moura, Nestor A. D. Mendes, Anderson M. Kayano, and Auro Nomizo for their collaboration of the work and manuscript.
This work was funded by the FIOCRUZ, FAPERO, Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Ministério da Ciência e Tecnologia (MCTI) from Brazil.
Availability of data and materials
The data sets used and/or analyzed during the current study are available from the authors (CSO, CASC, RDS, DLR, SM), upon reasonable request.
CSO, CASC, RDS, DLR and SM provided the biochemical and functional characterization. CSO, CASC, RDS, DLR, SM, LAM, ALF, CC, WLGC, MG, MDP and AMS conducted all the experiments, and analyzed and discussed the results obtained. JPZ, LAC, AMS, SM, DLR, MG and ALF participated in the analysis and discussion of the results, carried out a critical review of the work, and assisted in the writing and structuring of the article. AMS, DLR, MG and ALF were responsible for the conception of the work, supervised all the experiments and drafted the manuscript. All the authors read and approved the final manuscript.
Ethical approval and consent to participate
The procedures with animals are in accordance with the guidelines for the care and uses of laboratory animals from National Research Council, USA. The ethical aspects related to the project were approved by the Ethics Committee for Animal Use (No. 2012/1), (No. 102/2009) and Ethics Committee for Human Research – CEP from Brazil (CAAE: 14204413.5.0000.0011).
Consent for publication
The authors declare that they have no competing interests.
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